A Rho kinase (ROCK) inhibitor, fasudil, prevents matrix metalloproteinase-9-related hemorrhagic transformation in mice treated with tissue plasminogen activator.

Thrombolysis with tissue plasminogen activator (tPA) is the only FDA-approved therapy for acute ischemic stroke. However, hemorrhagic transformation, neurotoxicity, and a short treatment time window comprise major limitations for thrombolytic therapy. The purpose of the present study was to investigate whether fasudil, a Rho kinase (ROCK) inhibitor, would prevent tPA-associated hemorrhagic transformation and extend the reperfusion window in an experimental stroke model in mice. Mice subjected to 6-h middle cerebral artery occlusion were treated with delayed tPA alone, with combined tPA plus fasudil, or with a vehicle. We used histological and neurobehavioral measures to assess the effects of the treatment at 18 h and 7 days after the reperfusion. To investigate the mechanism of fasudil's beneficial effects further, we also performed an in vitro study with tPA and fasudil in human brain microvascular endothelial cells. Combination therapy with tPA plus fasudil prevented the development of hemorrhagic transformation, but did not reduce the infarct volumes. These changes significantly reduced mortality and increased locomotor activity at 7 days after the reperfusion. Furthermore, the administration of both drugs prevented injury to the human brain endothelial cells via the reduction of matrix metalloproteinase-9 (MMP-9) activity. These findings indicate that fasudil prevents the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tPA, at least in part, by inhibiting the increased activity of MMP-9 in endothelial cells.

A broad-spectrum matrix metalloproteinase inhibitor prevents hemorrhagic complications induced by tissue plasminogen activator in mice.

Delayed activation of tissue plasminogen activator (tPA) can lead to the disruption of the blood-brain barrier (BBB), resulting in hemorrhagic complications. In the present study, we focused on tight junction proteins (TJPs), occludin, zona occludens (ZO)-1, and claudin-5, which are important structural components of the BBB, and investigated whether inhibition of matrix metalloproteinases (MMPs) provides a protective effect against hemorrhagic complications induced by tPA. We subjected mice to 6-h filamental middle cerebral artery occlusion (MCAO) with vehicle, delayed tPA alone, or combined tPA (10 mg/kg, i.v.) plus GM6001 (100 mg/kg, i.p.), a broad-spectrum MMP inhibitor. We evaluated brain hemoglobin and the expression of MMP-9 and TJPs by immunoblotting. GM6001 significantly reduced tPA-elevated brain hemoglobin, MMP-9, and inhibited the degradation of occludin and ZO-1 induced by tPA, but not claudin-5. Treatment with GM6001 also significantly prevented the decrease in the survival rate and the reduction in locomotor activity caused by tPA at 7 days after ischemia/reperfusion. Furthermore, GM6001 treatment also significantly prevented cell damage, determined by release of lactase dehydrogenase (LDH) activity, and the decrease in transendothelial electrical resistance (TEER) induced by tPA. These findings indicate that GM6001 prevented the hemorrhagic complications and improved the behavioral abnormalities induced by tPA, partly via protection of TJPs. This suggests that GM6001 may be a useful candidate for combination therapy against the hemorrhagic complications induced by tPA.

Absence of paravascular nerve projection and cross-innervation in interscapular brown adipose tissues of mice.

To determine whether a paravascular nerve projection or cross-innervation exists in the interscapular brown adipose tissue (BAT), the distribution of noradrenergic or peptidergic nerve fibers in intact or denervated interscapular BAT pads of mice was examined histochemically. Noradrenaline (NA) fibers were visualized by the glyoxylic acid condensation method, and neuropeptide Y (NPY) and substance P (SP) fibers were detected immunohistochemically. Numerous NA-positive fibers and a few NPY- or SP-positive fibers were observed around intralobular arterioles in intact BAT pads. Some NA-positive fibers and very few NPY- or SP-positive fibers were seen around interlobular arteries. NA- and NPY-positive fibers were also found around brown adipose cells in the parenchyme of BAT, whereas SP-positive fibers were absent around the cells. However, all sections cut from denervated BAT pads of unilaterally or bilaterally denervated animals showed a total absence of NA-, NPY- or SP-positive nerve fibers. Therefore, neither a paravascular projection of NA, NPY, and SP fibers to the BAT nor a cross-innervation of these nerve fibers between the left and right BAT pads exists in the mouse BAT.